Cationic antimicrobial peptides (CAPs) are natural macromolecules whose broad-spectrum antibacterial activity involves interactions with phospholipids in bacterial membranes;this makes them a research subject of efforts supported by the National Institute of Allergy and Infectious Disease (NIAID) to explore and develop novel and more efficient therapeutics. However, attempts to produce synthetic CAPs for therapeutic use have been unsuccessful for the most part. This we feel is partially due to lack of a precise understanding of the specific consequences CAP interaction with the bacterial membrane has in gram- positive pathogens and how bacteria in turn respond to CAP activity. Research in this area will also yield a greater understanding of host-pathogen interactions, another research goal of the NIAID. Bacterial membranes contain localized microdomains of specific phospholipids involved in organizing protein components and in helping direct translocation of secreted elements. In Streptococcus pyogenes secretion of the major virulence factor SpeB occurs via the ExPortal, an anionic membrane microdomain enriched in secretory (Sec) translocons and the membrane anchored serine protease HtrA. The role of the ExPortal as a system coordinating protein secretion and maturation in S. pyogenes and the ability of cationic peptides to interact with charged membranes makes the ExPortal a likely target of the bactericidal action of these macromolecules. Preliminary data suggests that the action of the CAP Polymyxin B on S. pyogenes results in a disruption of ExPortal organization, leading to altered trafficking of streptococcal factors to their distinct membrane and post-secretion fates. Experiments are currently underway to test whether observations on polymyxin B interaction with the ExPortal extend to other physiologically relevant CAPs, the human defensins. I propose to utilize polymyxin B in a mutagenesis approach that will select for supressor mutations in gene products involved in ExPortal organization, function and antimicrobial resistance that will make streptococci resistant to disruption of the ExPortal by CAPs. The products of these mutated genes will be further characterized in terms of expression, localization, interaction with other proteins and role in pathogenesis. These approaches will yield novel information about how streptococci respond to CAPs, making this research relevant to public health given the enormous numbers treated for streptococcal disease annually and the ensuing potential for the appearance of drug resistance in these pathogens.